Procedure

Coat the glass bottom of a 35mm glass bottom dish with fibronectin (1:100 in PBS) for 1 hour

Seed 3k cells in up to 500ul of growth media onto the glass part of a small glass-bottom dish and maintain in culture in for 2 days

Cells will reach ~80% confluency

After the cells have adhered (1 hour after seeding) you may add another 1ml of growth media

Wash with warm PBS and add 1.5ml of serum starve media and culture for 3 days

Imaging

Microscope MP off; activate resonance scanner

Use the 100x oil objective

Argon: set to 25 to 30%

Image eGFP: excitation 488/emission 509

Set 488 laser to ~34%

Emission spectrum from: 500 to 600

Set Frequency: 512 x 512

Line Averaging: 4 is sufficient, 8 is really good

Use x, y, and z controls to focus on cells and find cilia.

Notes

Once you have focused on a cilium in xyz, it is best to zoom in to it so you can line up the plane of focus for imaging at xzy. Then switch to xzy. If the cilium is not parallel to the xz plane (dashed line), then rotate the field of view until it is parallel.

To image the entire cilium, make a xyz or xzy stack. It’s easiest to manually type in the z or y position, respectively, guessing at 0.1 um increments to find the edges of the cilium. Once you found one edge, click the begin arrow. Continue finding the other edge and then click the end arrow to complete the stack. A slice thickness of 0.1 to 0.2 um is ideal to capture the cilium (~400nm in diameter).